1. Field of the Invention
The present invention relates to a process for preparing DTPA derivatives. Particularly, the invention relates to a high yield process for the regiospecific synthesis of DTPA derivatives useful as bi-functional chelators in radioimmunotherapy and imaging.
2. Technology Background
There is an increasing interest in new radio-labeling agents for imaging, tumor detection and immuno therapy, which reduce the various disadvantages presented by conventional agents.
Conventional agents are generally based on radioactive halo-compounds, such as radioactive compounds based on iodine isotopes. However, these agents present a number of limitations. For example, the use of iodine isotopes is greatly limited by the high degradation rate of the carbon-iodine bond, in vivo. Another limitation relates to the less than ideal emission characteristics and physical half-lives of iodine radionuclides. Accordingly, there have been efforts to develop new tumor detection agents to overcome the shortcomings of halogen based radioactive labeling agents.
One avenue for providing more effective imaging and tumor detection agents is offered by organometallic compounds in the form of complex metallic radionuclides. Coupling the imaging and tumor detection agent to the protein is generally achieved through a covalent bond formed between the chelate and the protein by acylation with activated carbonyls, aromatic diazonium coupling, bromoacetyl alkylation or through thiourea bonds.
However, existing organometallic complexes do not provide optimum efficiency for radio labeling and therapy. For example, beyond the choice of a particular radioisotope, successful tumor detection and radioimmunotherapy, also depend on the selection of an effective chelate that joins easily to a particular antibody and allows for radionuclide insertion while preserving the antibody integrity.
Several factors must be considered in designing effective radiometal-chelated antibodies for imaging and tumor detection, and/or immunotherapy. For example, effective radionuclides must be selected according to their physical, chemical and biological properties. An optimal nuclide should be routinely available, easy to couple to the MAb and have an appropriate physical half-life to selectively detect and/or eliminate the target neoplastic tissue while sparing normal tissue.
The MAb that serves to carry the radionuclide to the tumor target must be selected based on the distribution of its antigenic target and on the specificity and binding affinity of the antibody to its target.
Another important aspect to consider in designing effective radiometal-chelated antibodies relates to the choice of the chelating agent (CA) used to couple the radionuclide to the antibodies. For example, effective radiometal-chelated antibodies must be stable in vivo. Stability in vivo depends on the condition that both the chelate linkage and the radiolabeling procedures not alter antibody specificity and bio-distribution.
In addition, selection and synthesis of the chelating agent is critical to optimize the adequacy between the chelate and the selected radionuclide and MAb. In particular, the choice and synthesis of the chelate should avoid inappropriate release of the radionuclide in vivo. This aspect is of paramount importance in that the most common problem associated with conventional chelating agents is their failure to link and securely hold to the antibody. As a consequence, there is considerable dissociation of the radionuclide in vivo from the MAb-CA complex prior to delivery of these agents to the surface of the tumor cell. Accumulation of free toxic radionuclides in normal tissue damages the normal tissue without the benefit of treating and/or detecting the tumor target. Another important aspect relates to the desirability that the selected chelating agent allow the MAb-CA complex to maintain the advantage provided by the specificity of the selected MAb.
Thus, several criteria must be considered in selecting adequate chelates for a selected MAb. For example, (a) addition of the CA should not alter the specificity or the binding affinity of the MAb to the antigenic target; (b) its addition to the MAb should not otherwise damage the antibody and thus alter its rates of catabolism or patterns of tissue distribution; (c) it should hold the radiometal tightly so that there is no premature elution of the radioisotiope from the MAb-CA complex in vivo; (d) linkage to the MAb should not alter the chelate's ability to retain the radionuclide; (e) the mode of linkage to the MAb should be as specific as possible to facilitate the design of protocols for the specific detection and therapy of tumor targets as well as the analysis of the data related to the detection of and treatment of the tumors; and (f) the chelate should be able to help clear the radionuclide following catabolism of the MAb-CA-radionuclide complex.
One group of suitable metal chelates is provided by diethylenetriaminepentaacetic acid (DTPA) and ethylendiaminetetraacetic acid (EDTA) and their derivatives. Chemically modified derivatives of (DTPA) and (EDTA) have been explored as metal ligands capable of effectively chelating radioactive metals, which can be easily coupled to immunoglobulins. However, these reagents were minimally effective due to the reduced affinity for the bound radionuclide, and consequent accumulation of radiochemical compounds in normal tissues.
A number of conventional methods are available for coupling EDTA and DTPA metal complexes to proteins. However, these methods have not reached the high efficiency rates required for effective imaging and tumor detection. For example, conventional methods present a number of disadvantages, such as the need for extensive purification prior to radiolabeling and the deficiency in chelating the metal, resulting from the use of a metal biding site in forming the covalent bond with the protein. Thus, new modes for protein linkage have been studied and new modes, which preserve all metal binding sites have been proposed.
For example, a detailed description of chemically modified ligands that would react rapidly and efficiently with antibody, and which retain the metal for a time that is long compared to the half-lives of radionuclides useful for imaging or therapy is provided by Brechbiel et al in "Synthesis of (1-(p-isothiocyanatobenzyl) derivatives of DTPA and EDTA. Antibody Labeling and Tumor-Imaging Studies." Inorg. Chem. 1986, 25, 2772-2781, the contents of which are incorporated herein by reference in their entirety.
Brechbiel et al propose EDTA and DTPA chemically modified chelates having an isothiocyanate group capable of efficient coupling to proteins. The synthesis of the chelates can be summarized as a "two-step process", wherein the first step includes generating an ethylendiamine or a diethylene triamine followed by alkylation of the amines to form the corresponding polyacetic acid, and the second step includes converting a functional group of the benzyl substitution to obtain a reactive moiety useful for protein coupling.
However, the above "two-step" process for the synthesis of MX-DTPA and the EDTA analog is limited to a low overall yield of less than 2%. The process requires tedious purification of intermediates, which include cation and anion exchange chromatography. In addition, the synthesis produces both regioisomers of MX-DTPA and has shown poor reproducibility.
Thus, there is a need for an alternative synthesis process for preparing DTPA derivatives with a high yield. It is desirable that the new synthesis eliminate the need for ion exchange chromatography in the separation of intermediates, thus providing a process that can be easily scaled up. Further, it is also desirable that such a process produce a single regioisomer of the chelate, thus allowing regiospecific synthesis of desired chelates useful as effective radiolabeling agents.